A comprehensive survey of protein-protein interactions between Plasmodium falciparum merozoites and human receptors

疟疾是世界上最常见的传染病之一，每年有3亿多疟疾病例，导致100多万人死亡，主要是非洲五岁以下儿童。疟疾是由单细胞疟原虫引起的，其中一种特别致命--恶性疟原虫--它是几乎所有疟疾死亡的罪魁祸首。疟原虫有一个复杂的生命周期，但疟疾的所有症状都是在它们入侵人类红细胞(也称为红细胞)时引起的。这种寄生虫利用红细胞作为食物来源，在红细胞内繁殖，48小时后，打开红细胞释放多种新的寄生虫。这种入侵、繁殖和再次入侵的循环导致了血液中大量的寄生虫。由于疟原虫在其生命周期的大部分时间都在人类细胞内度过，因此很难用疫苗针对它们，而且目前还没有获得许可的疟疾疫苗。然而，当寄生虫暴露在宿主免疫系统中时，红细胞入侵过程是寄生虫生命周期的少数阶段之一，因此是疫苗诱导的入侵阻断抗体或药物开发的潜在靶点。对于要开发的疫苗或药物，我们必须了解寄生虫表面的哪些蛋白质直接与红细胞结合，以及它们与之结合的红细胞蛋白质的特性。通过首先确定这些寄生虫与红细胞的相互作用，有可能开发出防止入侵的新疗法，从而治愈或预防疟疾。过去二十年的研究已经发现了许多可能与红细胞入侵有关的恶性疟原虫蛋白，但只有在极少数情况下，我们才知道它们与哪些红细胞蛋白结合。这在很大程度上是因为涉及的技术挑战：细胞表面之间的蛋白质-蛋白质相互作用通常非常短暂(只持续几秒钟)，因此很难使用标准的实验方法来检测。在威康信托桑格研究所，我们最近开发了一种新的方法来检测短暂的蛋白质-蛋白质相互作用。它被称为AVEXIS(基于Avidity的细胞外相互作用屏幕)，它使用在实验室表达和纯化的蛋白质文库来检测新的相互作用。在过去的两年里，我们已经开始将其应用于恶性疟原虫红细胞入侵的过程，并创建了红细胞和恶性疟原虫蛋白质的小型先导性文库，以筛选相互作用。这项初步工作发现了两种新的寄生虫-红细胞相互作用，其中一种似乎在红细胞入侵中发挥了非常重要的作用。然而，最初的筛选范围有限，我们仍然没有大多数恶性疟原虫蛋白的结合伙伴。在这项研究中，我们将扩大我们的寄生虫和红细胞蛋白质库，还包括恶性疟原虫可能与之相互作用的宿主血液的其他成分，如细胞表面存在的糖结构。在以All和All的方式筛选寄生虫和血液成分文库的相互作用后，我们将测试是否有任何新发现的相互作用在使用可以在实验室培养的恶性疟原虫入侵红细胞方面发挥作用。这项研究将建立恶性疟原虫蛋白质和人类血细胞成分之间相互作用的第一个系统图谱，并将确定对疫苗或药物开发特别感兴趣的蛋白质-蛋白质相互作用。我们还将把用于创建我们的重组蛋白库的DNA构建物存放在非营利性试剂资源库中，在那里其他研究人员可以自由访问它们，从而支持全球许多实验室的疟疾研究。

Malaria is one of the most common infectious diseases in the world, with more than 300 million cases of malaria each year, leading to more than 1 million deaths, primarily in children under the age of five in Africa. Malaria is caused by single-celled Plasmodium parasites and one species is particularly deadly - Plasmodium falciparum - which is responsible for almost all malaria deaths. Plasmodium parasites have a complex life cycle, but all the symptoms of malaria are caused when they invade human red blood cells, also known as erythrocytes. The parasite uses the erythrocyte as a source of food, multiplies inside it, and, after 48 hours, breaks open the erythrocyte to release multiple new parasites. This cycle of invasion, multiplication and re-invasion results in high numbers of parasites in the blood stream.Because Plasmodium parasites spend the majority of their life cycle inside human cells, they are difficult to target with vaccines, and there is currently no licensed vaccine for malaria. However, the process of erythrocyte invasion is one of the few stages of the parasite's life cycle when they are exposed to the host immune system, and is therefore a potential target for vaccine-induced invasion-blocking antibodies or drug development. For vaccines or drugs to be developed, it is critical that we understand which proteins on the parasite surface are binding directly to erythrocytes and the identity of the erythrocyte proteins that they are binding to. By first identifying these parasite-erythrocyte interactions, it may be possible to develop novel therapeutics that prevent invasion and hence cure or prevent malaria. Research over the last two decades has led to the identification of numerous P. falciparum proteins that may be involved in erythrocyte invasion, but only in very cases do we know which erythrocyte proteins they bind to. This is in large part because of the technical challenges involved: protein-protein interactions between the surface of cells are often very short-lived (lasting only a few seconds) and so are very difficult to detect using standard experimental approaches.At the Wellcome Trust Sanger Institute we have recently developed a new approach to detect short-lived protein-protein interactions. Called AVEXIS (Avidity based Extracellular Interaction Screen), it uses libraries of proteins, expressed and purified in the lab, to detect novel interactions. Over the last two years we have started to apply this to the process of P. falciparum erythrocyte invasion and have created small pilot libraries of erythrocyte and P. falciparum proteins to screen for interactions. This preliminary work has identified two new parasite-erythrocyte interactions, one of which appears to play a very important role in erythrocyte invasion. However, this initial screen was limited in scope and we still do not have binding partners for the majority of P. falciparum proteins. In this research we will expand our libraries of parasite and erythrocyte proteins, and also include other components of the host blood which P. falciparum parasites may be interacting with, such as sugar structures that are present on cell surfaces. After screening the parasite and blood component libraries for interactions in an all vs. all manner, we will test whether any newly discovered interactions have roles in erythrocyte invasion using P. falciparum parasites that can be cultured in the lab. This research will build up the first systematic map of interactions between P. falciparum proteins and human blood cell components, and will identify protein-protein interactions that are of particular interest for vaccine or drug development. We will also deposit the DNA constructs that are used to create our recombinant protein libraries in not-for-profit reagent resource collections, where they can be freely accessed by other researchers and thereby empower malaria research in many labs across the globe.